Iron is required by most organisms for survival and plays a role in many biological processes including oxygen and electron transport, nitrogen fixation, and DNA synthesis. Free iron is toxic to cells due to its ability to form reactive hydroxyls that cause peroxidation of lipid membranes and damage DNA. Cells maintain iron homeostasis by tightly regulating the amount of iron taken up by cells and the amount of iron stored in cells. Iron homeostasis is regulated by the iron-regulatory proteins (IRPs). Iron regulatory proteins 1 and 2 (IRP1 and 2) are key proteins involved in cellular iron homeostasis. IRP1 and IRP2 are cytosolic RNA binding proteins that regulate the translation or the stability of mRNAs encoding proteins involved in uptake, sequestration and utilization of iron. IRP1 and IRP2 bind to specific stem-loop structures, termed the Iron-responsive elements (IREs), located in either the 5'- or 3'-untranslated regions of specific mRNAs. The binding of IRPs to 5' IREs results in translational repression, while the binding of IRPs to 3' IREs results in mRNA stabilization. Although IRP1 and IRP2 regulate some of the same IRE- mRNAs, they have preferences for specific IRE-mRNAs. IRP1 exhibits two mutually exclusive activities depending on cellular iron levels: When iron is scarce, IRP1 binds to IREs, regulating mRNA translation or stability; when iron is abundant, IRP 1 exhibits aconitase activity, catalyzing the interconversion of citrate and isocitrate. Unlike IRP 1, IRP2 lacks aconitase activity and it is regulated by iron by proteolysis. In addition to iron, IRPs are regulated by hypoxia, indicating that they have important roles in the regulation of iron homeostasis during low oxygen conditions. We plan to address the roles of the two IRPs in regulating cellular iron metabolism. The specific aims of this proposal l) to isolate novel IRE mRNAs using a functional genomics screening approach, 2) to determine the mechanisms altering iron homeostasis in aconitase transgenic mice and in cultured cells, and 3) to determine the mechanisms regulating IRP1 and IRP2 during hypoxia.